Peritoneal membrane infiltration of macrophages and expression of macrophage-related chemokines.

<p><b>(A-C)</b> Representative photomicrographs of immunohistochemistry for macrophages (ED1⁺) (x200). <b>(A):</b> Only few ED1⁺ cells were present in the normal peritoneal membrane; <b>(B)</b> PF group showed a marked increase in the number of macrophages infiltrating peritoneal membrane; <b>(C)</b> Animals with PF treated with VPA exhibited a non-significant reduction of macrophage infiltration. <b>(D)</b> Quantitative analysis of the number of macrophages of all groups (n = 5/group). <b>(E-F)</b> Concentration of MCP-1 and MIP-2 in the peritoneal tissue, detected by Multiplex. VPA treatment reduced the protein levels of macrophage chemoattractant proteins (n = 4-5/group).</p

Representative photomicrographs of parameters related to experimental peritoneal fibrosis and effect of treatment with VPA (x200).

<p><b>(A-C)</b> Effect of VPA on the peritoneal expression of α-SMA (myofibroblasts) by immunohistochemistry. <b>(A):</b> No α-SMA expression was detected in the peritoneal membrane of the Control group. <b>(B)</b> Induction of PF was associated with a marked increase in α-SMA expression. <b>(C)</b> A striking reduction of myofibroblasts was observed with VPA treatment. <b>(D-F)</b> Effect of VPA on the peritoneal expression of pSmad3, by immunohistochemistry. <b>(D)</b> Only a few positive cells were noted in the peritoneal membrane of the Control group; <b>(E)</b> PF group showed an increased number of pSmad3 positive cells (stained in brown) infiltrating peritoneal membrane; <b>(F)</b> pSmad3 expression was significantly reduced by VPA treatment. <b>(G-H)</b> Quantification analysis of the effect of VPA in experimental PF, regarding α-SMA staining area, and the number of pSmad3 positive cells in all groups (n = 5-6/group). <b>(I)</b> Quantification analysis of the effect of VPA in the peritoneal membrane expression of mRNA Smad3 in all groups (n = 6/group).</p

Capillary density and VEGF mRNA expression in the peritoneum.

<p><b>(A-C)</b> Representative immunofluorescence photomicrographs of Isolectin-B₄ (stained in red), an endothelium marker, and DAPI+ cells (stained in blue) in the peritoneal membrane. While virtually no vessels were identified in Control Group <b>(A),</b> an intense neoangiogenesis (white arrows) in the submesothelial zone was observed in the PF group <b>(B)</b>. <b>(C)</b> VPA treatment attenuated this increase. <b>(D)</b> Graph comparing the vascular density of all groups(n = 3-4/group). <b>(E)</b> Expression of mRNA VEGF in peritoneal tissue by PCR (n = 6/group).</p

Expression of fibronectin, TGF-β, FSP-1, and BMP-7 genes in the peritoneal tissue by qPCR.

<p>VPA treatment reduced the upregulation of the profibrotic genes observed in the PF group and increased BMP-7 expression depressed in the PF group (n = 6/group).</p

Anti-fibrotic effects of valproic acid in experimental peritoneal fibrosis - Fig 1

<p><b>Effect of VPA on the peritoneal thickness in experimental PF model (A-C):</b> Representative photomicrographs of peritoneal samples stained with Masson’s trichrome (x200)<b>. (A)</b> Control group (n = 8) showed a thin submesothelial layer of the peritoneum, without any morphological changes. <b>(B)</b> The peritoneal membrane in the PF group (n = 10) rats showed a marked thickening of the submesothelial compact zone. <b>(C):</b> VPA prevented the development of submesothelial thickening in the PF+VPA group (n = 10). <b>(D)</b> Quantification analysis of the effect of VPA on peritoneal thickness, at day 30.</p

Expression of proinflammatory cytokines in the peritoneal membrane of the different groups.

<p>VPA treated animals showed a significant lower gene (n = 6/group) and protein expression (n = 4-5/group) of TNF-α in peritoneal tissue than the PF group.</p